Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany
Sindhuja Gowrisankaran
Synaptic Vesicle Dynamics Group, European Neuroscience Institute, University Medical Center Göttingen, Göttingen, Germany
Matija Krunic
Synaptic Vesicle Dynamics Group, European Neuroscience Institute, University Medical Center Göttingen, Göttingen, Germany
Burkhard Rammner
Sciloop, Hamburg, Germany
Andrew Woehler
Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany
Eileen M Lafer
Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, United States; Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, United States
Carsten Mim
Department for Biomedical Engineering and Health Solutions, Kungliga Tekniska Högskolan, Huddinge, Sweden; Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden
Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
